I am going to perform an ELISA to measure dopamine release from neurons differentiated from stem cells. I am going to use 56mM KCl on the cells for 15 minutes then collect the media and add 0.1mM EDTA to stabilise released dopamine. However, my protocol is not clear what I dilute the KCl in - ie when the KCl is added to the cells, is it PBS or media or maybe HBSS?
Simply diluting KCl in your medium is not a good idea. In this case you would raise the osmolarity from approx. 300 mOsm to 400 mOsm. Even if you use controls as those mentioned above this is quite a stress for the cells. Instead you should substitute Na+ in your medium with K+, e.g. use 50 mM less NaCl and add 50 mM KCl to raise K+ form 3 to 53 mM. (The actual number will depend on your medium formula, usually they allready contain 3-5 mM K+.) If you use readymade medium for your experiment, you might prepare ACSF for stimulation period. With normal and high K+ as described above. BTW Another possibility to stimulate DA release is the application of 1-5 mM 4-aminopyridine (4-AP). This tiny amount can be diluted directly in any medium without changing the osmolarity too much.
I would suggest to use the medium your cells are in, because otherwise you change not only KCl but osmolarity too.
Also check separately, if possible, if NaCl or Mannitol do the same as KCl, maybe your dopamine release is not due to depolarisation but to changes in osmolarity.
I agree with Erik. It's best to use the medium that your cells are in as the diluent.
Simply diluting KCl in your medium is not a good idea. In this case you would raise the osmolarity from approx. 300 mOsm to 400 mOsm. Even if you use controls as those mentioned above this is quite a stress for the cells. Instead you should substitute Na+ in your medium with K+, e.g. use 50 mM less NaCl and add 50 mM KCl to raise K+ form 3 to 53 mM. (The actual number will depend on your medium formula, usually they allready contain 3-5 mM K+.) If you use readymade medium for your experiment, you might prepare ACSF for stimulation period. With normal and high K+ as described above. BTW Another possibility to stimulate DA release is the application of 1-5 mM 4-aminopyridine (4-AP). This tiny amount can be diluted directly in any medium without changing the osmolarity too much.
I agree with Torsten. Just adding KCl will rise osmolarity. 4-AP is also a good idea.
Since you measure the dopamine release after a 15 minute KCl stimulation, I hope you are not going to re use your neurons for further experiments. In that case, it is best to use HEPES buffered Artifical cerebrospinal fluid (ACSF) instead of your growth media for experiments. The composition is NaCl 89 mM , KCl 56 mM, NaHCO3 5 mM, NaH2P04 1.2 mM, MgCl2 1 mM, Glucose 10 mM, HEPES 10 mM, CaCl2 dihydrate 1.33 mM or 2 mM. Also note that the concentration of Calicum in your ACSF will influence dopamine release from your neurons.
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